Inward rectifying potassium (Kir) channels regulate excitability in many tissues, and multiple diseases result from mutations of Kir channel genes. The long-term goal of this project is to understand the molecular details of Kir channel function. Previously, we discovered that soluble cytoplasmic polyamines cause inward rectification and demonstrated their mechanism and sites of action in the channel. We have developed novel systems for large-scale purification of bacterial and human Kir channels, and have succeeded in functional analysis of these recombinant channel proteins in reconstituted membrane systems. Together with ideas generated by recent crystal structures of both pro- and eukaryotic Kir channels, our novel approaches to biochemical and functional analysis of these channels allow us to develop and address exciting new questions and hypotheses regarding the fundamental basis of Kir channel activity. We will determine the molecular mechanisms by which lipids regulate gating in model Kir channels, and the dynamic structural changes that accompany gating, by combinations of biochemical and electrophysiological recordings.